Electroporation Transformation - Revision history

Macampbell at 13:46, 21 January 2018

2018-01-21T13:46:46Z

Macampbell at 21:59, 27 June 2016

2016-06-27T21:59:59Z

Ekeffeler at 19:40, 10 July 2014

2014-07-10T19:40:06Z

Eckdahl at 20:58, 7 July 2014

2014-07-07T20:58:03Z

Eckdahl at 18:56, 7 July 2014

2014-07-07T18:56:24Z

Eckdahl at 15:42, 10 March 2014

2014-03-10T15:42:35Z

Eckdahl at 14:39, 10 March 2014

2014-03-10T14:39:13Z

Eckdahl at 14:38, 10 March 2014

2014-03-10T14:38:52Z

Eckdahl at 14:37, 10 March 2014

2014-03-10T14:37:21Z

Eckdahl: Created page with 'This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA. '''Cell Preparation''' 1. This procedure yields enough bacteria for two transformati…'

2014-03-10T14:36:32Z

Created page with 'This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA. '''Cell Preparation''' 1. This procedure yields enough bacteria for two transformati…'

New page

This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA.

'''Cell Preparation'''

1. This procedure yields enough bacteria for two transformations. Scale up for more.
2. Grow bacteria to be transformed in 40 ml of broth in a 50 ml tuble, allowing them to grow to an OD590 of 0.5 to 1.0 (optional: just grow overnight)
3. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH2O
4. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O
5. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O
6. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH2O
7. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 80 ul sterile 10% glycerol (optional: 72 ul sterile dH2O and 8 ul sterile glycerol)

'''Electroporation'''

1. Set charging voltage to 1.3 - 1.5 kV
2. Pipette 1 ul of plasmid (1 ng/ul) into the electroporation cuvette
3. Add 40 ul of electrocompetent bacteria to the cuvette and flick gently
4. Click the Pulse button
5. Transfer to sterile 1.5 ml tube and immediately add 960 ul sterile LB or SOC
6. Incubate at 37 C with shaking for 1 hour
7. Spin in microfuge for 30 seconds
8. Add 50 ul LB + antibiotic and spread onto agar plate with antibiotic

'''Washing Electroporation Cuvettes'''

1. Fill the cuvettes two times with dH2O, dumping each time
2. Fill the cuvettes with 0.1 N NaOH and let sit for 5 minutes
3. Fill the cuvettes two times with dH2O, dumping each time
4. Fill the cuvettes two times with 95% ethanol, dumping each time
5. Turn upside down to dry

@@ Line 31: / Line 31: @@
 .	Incubate at 37 C with shaking for 1 hour
-.	Spin in microfuge for 60 seconds, dump off supernatant
+.	Spin in microfuge for 60 seconds, pour off all supernatant
-.	Add 50 ul LB + antibiotic, resuspend supernatant, and spread onto agar plate with antibiotic
+.	Add 50 ul LB + antibiotic, resuspend pellet, and spread onto agar plate with antibiotic

@@ Line 14: / Line 14: @@
 .	Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH2O
-.	Transfer to a sterile 1.5 mL tube and pellet the culture by centrifuging in a microfuge for 1 minute; resuspend in 80 ul sterile 10% glycerol (optional: 72 ul sterile dH2O and 8 ul sterile glycerol)
+.	Transfer to a sterile 1.5 mL tube and pellet the culture by centrifuging in a microfuge for 1 minute; resuspend in 80 ul sterile 10% glycerol
@@ Line 31: / Line 31: @@
 .	Incubate at 37 C with shaking for 1 hour
-.	Spin in microfuge for 30 seconds
+.	Spin in microfuge for 60 seconds, dump off supernatant
-.	Add 50 ul LB + antibiotic and spread onto agar plate with antibiotic
+.	Add 50 ul LB + antibiotic, resuspend supernatant, and spread onto agar plate with antibiotic

← Older revision		Revision as of 14:39, 10 March 2014
Line 1:		Line 1:
−	This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA.	+	'''This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA'''
		+

	'''Cell Preparation'''		'''Cell Preparation'''

@@ Line 8: / Line 8: @@
 .	Grow bacteria to be transformed in 40 ml of broth (eg. LB Broth) in a 50 ml tuble, allowing them to grow to an OD590 of 0.5 to 1.0 (optional: just grow overnight)
-.	Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH2O
+.	Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH<sub>2</sub>O
-.	Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O
+.	Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH<sub>2</sub>O
-.	Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH2O
+.	Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH<sub>2</sub>O
 .	Transfer to a sterile 1.5 mL tube and pellet the culture by centrifuging in a microfuge for 1 minute; resuspend in 80 ul sterile 10% glycerol
@@ Line 39: / Line 39: @@
 '''Washing Electroporation Cuvettes'''
-.	Fill the cuvettes two times with dH2O, dumping each time
+.	Fill the cuvettes two times with dH<sub>2</sub>O, dumping each time
 .	Fill the cuvettes with 0.1 N NaOH and let sit for 5 minutes
-.	Fill the cuvettes two times with dH2O, dumping each time
+.	Fill the cuvettes two times with dH<sub>2</sub>O, dumping each time
 .	Fill the cuvettes two times with 95% ethanol, dumping each time
 .	Turn upside down to dry

@@ Line 25: / Line 25: @@
 .	Add 40 ul of electrocompetent bacteria to the cuvette and flick gently
-.	Click the Pulse button
+.	Click the Pulse button. If there is too much salt, the cuvette can arch and make a pop sound. [https://vimeo.com/user37250198/review/172478413/c3537bdf0e see example]
 .	Transfer to sterile 1.5 ml tube and immediately add 960 ul sterile LB or SOC

@@ Line 21: / Line 21: @@
 '''Electroporation'''
-.	Set charging voltage to 1.3 - 1.5 kV
+.	Use a clean electroporation cuvette with chamber gap of 1 mm and set charging voltage to 1.3 - 1.5 kV
 .	Pipette 1 ul of plasmid (1 ng/ul) into the electroporation cuvette